Hepatitis C virus (HCV) is a major cause of chronic hepatitis, with complications of liver failure and cancer, throughout the world. Prospects for developing an effective vaccine remain unlikely, and the development of new antivirals has been limited by the lack of model HCV systems. The further characterization of selected populations of patients with chronic HCV, and the development of model systems needed to study HCV replication and to identify molecular-based therapeutics and the development of model systems needed to study HCV replication and to identify molecular-based therapeutics represents major unsolved problems. The HCV RNA-dependent RNA polymerase (RDRP) is a likely target for HCV antivirals. We have expressed recombinant HCV RDRP an isolated it as an active enzyme. In addition, we have detected circulating antibodies to recombinant HCV RDRP in some patients - this may provide another index of viral replication. Moreover, we have made progress in developing a system that propagates HCV in cultured cells. The hypothesis to be tested is: that model in vitro and cellular systems can be developed to study key aspects of HCV replication, and to identify molecular-based therapeutics that directly target the HCV RNA-dependent RNA polymerase. Specific Objectives: 1) to purify and characterize recombinant HCV RNA-dependent RNA polymerase (RDRP) that we are expressing in E. coli and have isolated in an active form; 2) to use our HCV RDRP assay to identify small molecules that directly inhibit this enzyme. 3. To use purified HCV RDRP to perform immunoassays that will be used to further characterize patients with chronic hepatitis C, and 4) to do feasibility studies for future crystal growth work. The use the gene that expresses active HCV RNA=dependent RA polymerase to develop model cellular systems that will permit further studies of this enzyme and identify small molecules that enter intact cells and inhibit the HCV RDRP. To continue studies on a promising cell culture system that propagates HCV mouse studies. Our long-range goal will be to use this system to study details of viral gene expression and replication, and to identify moved HCV antiviral agents.